Biomanufacturing suite and methods for large-scale production of cells, viruses, and biomolecules

ABSTRACT

The present invention provides a production module for large-scale production of cells and/or cell-derived products such as antibodies or virus; a production suite comprising a plurality of functionally connected production modules of the invention; and a method for large-scale production of cells and/or cell-derived products using the production modules and/or production suites of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is the National Stage of InternationalApplication No. PCT/US2013/057156, filed Aug. 28, 2013, which claims thebenefit of U.S. Provisional Application Ser. No. 61/694,184, filed Aug.28, 2012, each of which is hereby incorporated by reference herein inits entirety, including any figures, tables, nucleic acid sequences,amino acid sequences, or drawings.

BACKGROUND OF THE INVENTION

The anticipated growth of cell production, virus production, and cellculture-based biomolecule production will require new paradigms forrapid, high-throughput, harvest, purification, concentration andformulation of a variety of cell, viruses, and biomolecules such asproteins and immunoglobulins. Manual methods for producing and purifyingthese products have their drawbacks. For example, these methods can belabor intensive, time consuming, and are highly inefficient. Large scalemanufacturing techniques typically use multiple columns which aremanually packed with resin and sterilized prior to each purificationrun. The manual steps involved in these methods also include a high riskof contamination. Moreover, conventional approaches and tools for theseproducts typically involve numerous manual manipulations that aresubject to variations even when conducted by skilled technicians. Whenused at the scale needed to manufacture hundreds or thousands ofdifferent cells, cell lines and patient-specific cell based therapies,for example, the variability, error or contamination rate may becomeunacceptable for commercial processes.

One type of method for the production of cell-secreted products is touse a bioreactor (e.g., hollow fiber, ceramic matrix, fluidizer bed,etc.) in lieu of the stirred tank. This can bring facilities costs downand increases product concentration. The systems currently available aregeneral purpose in nature and require considerable time from trainedoperators to setup, load, flush, inoculate, run, harvest, and unload.Each step typically requires manual documentation, which is laborintensive and subject to errors.

Cell culturing devices or cultureware for culturing cells in vitro areknown. Hollow fiber perfusion bioreactors (HFBx) were first introducedin 1972 as a model system to study tumors growing at tissue-likedensities (R. A. Knazek et al., Science, 1972, 178(56):65-67). Thissystem is a high-density, continuous-perfusion system cell culturesystem that has been used for the production of secreted proteins suchas hybridoma, Chinese hamster ovary cells (CHO), human embryonic kidney(HEK) 293 cells, and other mammalian and insect cells. HFBx have beenused in a variety of applications such as bioartificial organs,pharmacokinetics, cell therapy, toxicology, etc. In the mid-1980s,Biovest International (formerly Endotronics, Inc.) developed the firstcommercial scale HFBx system and ever since, the most common applicationfor this technology has been the large-scale production of mammaliancell-secreted proteins, predominantly monoclonal antibodies.

As disclosed in U.S. Pat. No. 4,804,628, the entirety of which is herebyincorporated by reference, a hollow fiber culture device includes aplurality of hollow fiber membranes. Medium containing oxygen,nutrients, and other chemical stimuli is transported through the lumenof the hollow fiber membranes or capillaries and diffuses through thewalls thereof into an extracapillary (EC) space between the membranesand the shell of the cartridge containing the hollow fibers. The cellsthat are to be maintained collect in the EC space. Metabolic wastes areremoved from the bioreactor. The cells or cell products can be harvestedfrom the device.

There is a need for a system and method whereby cells and cell-derivedproducts can be produced on a large-scale in an automated, rapid andsterile manner.

BRIEF SUMMARY OF THE INVENTION

An aspect of the invention provides a production module for large-scaleproduction of cells and/or cell-derived products, comprising:

-   -   (a) a cultureware module comprising one or more bioreactors        (cell growth chambers) and an interface plate (cultureware        support structure) with the one or more bioreactors mounted        thereto; and    -   (b) an instrument module comprising hardware to support cell        culture growth, wherein the instrument module and the        cultureware module are adapted for removable attachment to one        another.

In some embodiments, cultureware module includes a plurality ofbioreactors. For example, each cultureware module may have 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or more bioreactors. In some embodiments, thebioreactors are hollow-fiber bioreactors.

Another aspect of the invention concerns production suite comprising aplurality of production modules of the invention, functionallyconnected. The production suite may further include a room for enclosingthe plurality of production modules and having one or more supportsurfaces for supporting the plurality of production modules. Preferably,the room is environment-controlled (e.g., temperature-controlled) toachieve the desired cell culture conditions. Consequently, no furtherheat/cooling source is required locally at the site of thebioreactor(s). In some embodiments, the room is a modular and/orrelocatable building. For example, the building may have one or morereceivers affixed to the building frame or wall, for receiving a liftingattachment allowing transport of the building onto a truck, trailer,vessel, aircraft, or other conveyance.

Another aspect of the invention is a method for large-scale productionof cells and/or cell-derived products, comprising providing one or moreproduction modules or one or more production suites of any precedingclaim, introducing cells into the one or more bioreactors; culturing thecells to produce grown cells and/or cell-derived products; andharvesting the grown cells and/or cell-derived products. Any desiredcell type may be used, e.g., mammalian cells, insect cells, avian cells,or plant cells. Any desired cell-derived product may be produced if asatisfactory cell type(s) is available, such as immunoglobulins,proteins, viruses, and virus-like particles. In one embodiment, themethod for large-scale production cells and/or cell-derived productscomprises:

providing at least one production module, the production modulecomprising: (a) a cultureware module comprising one or more bioreactorsand an interface plate with the one or more bioreactors mounted thereto,and (b) an instrument module comprising hardware to support cell culturegrowth, wherein the instrument module and the cultureware module areadapted for removable attachment to one another;

introducing cells into the cell growth chamber;

fluidly attaching a source of cell culture medium to the culturewaremodule;

operating the pump to circulate the cell culture medium through thebioreactor to grow cells therein; and

collecting the grown cells or cell-derived products, and optionallypurifying the cells or cell-derived products.

The method may further include removably attaching the production moduleto the instrument module prior to introduction of the cells. The methodmay include programming operating parameters into a microprocessorcontrol. In some embodiments, cells are introduced into bioreactors ofmultiple production modules as a production suite, and grown cells orcell-derived products are collected from the multiple productionmodules.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a production suite of the invention, with48 production modules (a cultureware module having 10 bioreactorsinstalled on the interface plate is shown in the inset).

FIG. 2 shows an embodiment of a production module of the invention,including an instrument module and a cultureware module including thefront surface of an interface plate and a series of bioreactors betweenthe automated valves.

FIG. 3 shows an embodiment of a cultureware module of the invention,showing the rear surface of the interface plate; extracapillary (EC)reservoir and intracapillary (IC) reservoir, which connect toappropriate manifolds for the bioreactor(s) as exemplified in the flowpath schematic of FIG. 5; and a gas exchanger connected to one or morebioreactors and the IC reservoir.

FIG. 4 shows an example of a cultureware flow paths schematic of theinvention showing connections between production modules (inter-moduleconnections).

FIG. 5 shows an example of a cultureware module flow path schematic ofthe invention. Inoculum such as cells and viruses can be introduced asindicated. The schematic includes a pre-bioreactor sensor array (lowersensor array in figure) and post-bioreactor sensor array (upper sensorarray in figure), for sensing one or more parameters such as pH,dissolved oxygen, temperature, pressure, and, optionally, variousmetabolites. FIG. 5 also shows an online intercapillary metabolicanalyzer for sampling fluid and measuring parameters such as lactate andCO₂.

FIG. 6 shows an embodiment of a cultureware module and instrument moduleof the invention. Together, the cultureware module and instrument moduleare referred to as a production module.

FIG. 7 shows an embodiment of a hollow fiber perfusion bioreactor whichmay be used in the production modules, production suites, and methods ofthe invention, with hollow fibers and in and out ports for theintracapillary space and extracapillary space indicated. As will beappreciated by those skilled in the art, the sidedness and orientationof the ports on the bioreactor are not critical.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a production module, production suite,and method for the large-scale production of cells and cell-derivedproducts, such as proteins, antibodies, virus and virus like particles.The production suite includes multiple instrument modules, preferablysituated in a controlled-environment (e.g., temperature, and/orhumidity) room. Each instrument module supports a one-time-usedisposable cultureware module. The modules have local independentcontrols to support the cultureware module and are networked to allowcoordination between modules or automated analyzers. The modules arerepeated to provide a variable large scale production suite. As such,the production suite can start with a small number of instrument modulesand be added to as production need increases. The instrument modules canbe “hot swapped” so that a module can be removed for maintenance withoutinterfering with the other modules in the suite. The room enclosing themodules is temperature controlled for the culture requirements. Theproduction suite will also have environmental filtration and containmentnecessary for the product produced. Controlled ingress and egress willbe to a gowning area. In order to minimize the number of times theoperator needs to gown and enter the room, external control andmonitoring stations are provided to allow the operator to check or makeadjustments. Video monitoring of the modules may be achieved using videosurveillance equipment in the normal visual spectrum, as well as nightvision for dark or low light settings.

The cultureware modules will allow ease and quickness of setup without-of-the-package installation. This will eliminate the need for abiological hood to make aseptic connections. Connection at thecultureware module will be made with sterile fluid connectors or steriletubing splicer. Fluid sensing elements will come sterile in thecultureware module. Mechanical interfacing will occur when thecultureware module is inserted into the instrument module. Predefinedautomated procedures will fluidically prepare the cultureware module.Since a variety of different cell and viral types are anticipated,flexibility of inoculation is important. Support for bag, spinner orinter-cultureware module inoculations will be supported. This will allowsuspension, attachment dependent or primary cells (such as stem cells)to be used as well as various viral (free virus, infected cells, etc.)infectious agents. The figures show hollow-fiber bioreactors being usedto support the culture but other bioreactors (such as synthetic orbiological based scaffolding, immobilization gel, etc.) may be useddepending on the culture requirements. Harvesting of product (such asexpressed protein, viruses, viral particles, or cells) is equallyimportant. Harvest can be from an individual cultureware module to acontainer, a group of modules to a container (batch harvest), or use ofthe harvest (individual or batch) to inoculated other culturewaremodules. Online monitoring of the cultureware module will be done at theinstrument module using the pre-sterile sensing elements (such as pH ordissolved oxygen) or instrument module sensors (such as gas pressure,mass flow, fluid flow or off-gas CO₂). The cultureware module willsupport offline analysis for sensor calibration, metabolic analysis andcell/product analysis.

One aspect of the invention provides a production module for large-scaleproduction of cells and/or cell-derived products, comprising:

-   -   (a) a cultureware module comprising one or more bioreactors        (cell growth chambers) and an interface plate (cultureware        support structure) with the one or more bioreactors mounted        thereto; and    -   (b) an instrument module comprising hardware to support cell        culture growth, wherein the instrument module and the        cultureware module are adapted for removable attachment to one        another.

In some embodiments, cultureware module includes a plurality ofbioreactors. For example, each cultureware module may have 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or more bioreactors. In some embodiments, thebioreactors are hollow-fiber bioreactors.

Another aspect of the invention concerns production suite comprising aplurality of production modules of the invention, functionallyconnected. The production suite may further include a room for enclosingthe plurality of production modules and having one or more supportsurfaces for supporting the plurality of production modules. Preferably,the room is environment-controlled (e.g., temperature-controlled) toachieve the desired cell culture conditions. Consequently, no furtherheat/cooling source is required locally at the site of thebioreactor(s). In some embodiments, the room is a modular and/orrelocatable building. For example, the building may have one or morereceivers affixed to the building frame or wall, for receiving a liftingattachment allowing transport of the building onto a truck, trailer,vessel, aircraft, or other conveyance.

Another aspect of the invention is a method for large-scale productionof cells and/or cell-derived products, comprising providing one or moreproduction modules or one or more production suites of any precedingclaim, introducing cells into the one or more bioreactors; culturing thecells to produce grown cells and/or cell-derived products; andharvesting the grown cells and/or cell-derived products. Any desiredcell type may be used, e.g., mammalian cells, insect cells, avian cells,or plant cells. Any desired cell-derived product may be produced if asatisfactory cell type(s) is available, such as immunoglobulins,proteins, viruses, and virus-like particles. In one embodiment, themethod for large-scale production cells and/or cell-derived productscomprises:

providing at least one production module, the production modulecomprising: (a) a cultureware module comprising one or more bioreactorsand an interface plate with the one or more bioreactors mounted thereto,and (b) an instrument module comprising hardware to support cell culturegrowth, wherein the instrument module and the cultureware module areadapted for removable attachment to one another;

introducing cells into the cell growth chamber;

fluidly attaching a source of cell culture medium to the culturewaremodule;

operating the pump to circulate the cell culture medium through thebioreactor to grow cells therein; and

collecting the grown cells or cell-derived products, and optionallypurifying the cells or cell-derived products.

The method may further include removably attaching the production moduleto the instrument module prior to introduction of the cells. The methodmay include programming operating parameters into a microprocessorcontrol. In some embodiments, cells are introduced into bioreactors ofmultiple production modules as a production suite, and grown cells orcell-derived products are collected from the multiple productionmodules.

Some examples of applications for which the production modules,production suites, and methods of the present invention include, but arenot limited to:

-   -   The production of monoclonal antibodies from hybridoma cell        lines (e.g., the K6H6/B5 or 1D12 hybridoma cell lines).    -   The expansion of autologous patient-derived blood cells        including immune cells for therapeutic application.    -   The expansion of patient derived somatic cells for subsequent        re-infusion back into patients for therapeutic purposes. A        specific example already available for therapeutic application        in patients is the harvesting and expansion of patient-specific        cartilage cells (chondrocytes) followed by re-infusion of those        cells back into a region containing damaged articular cartilage.    -   The expansion of patient-derived or generic multipotent cells,        including embryonic stem cells, adult stem cells, hematopoeitic        stem or progenitor cells, multi- or pluripotent cells derived        from cord blood or other sources for therapeutic purposes.    -   The expansion of somatic or germline cells as in the        aforementioned cellular applications and in which the cells have        been genetically modified to express cellular components or to        confer on them other beneficial properties such as receptors,        altered growth characteristics or genetic features, followed by        introduction of the cells into a patient for therapeutic        benefit. An example is the expansion of patient specific        fibroblasts genetically modified to express growth factors,        clotting factors, or other biologically active agents to correct        inherited or acquired deficiencies of such factors.    -   The production of virus (such as influenza), VLPs, and viral        vectors, e.g., for production of vaccines.    -   The production of other cell-derived products such as growth        factors.

I. PRODUCTION MODULES

Accordingly, an aspect of the invention provides a production module forlarge-scale production of cells and/or cell-derived products,comprising:

-   -   (a) a cultureware module comprising one or more bioreactors        (cell growth chambers) and an interface plate with the one or        more bioreactors mounted thereto; and    -   (b) an instrument module comprising hardware to support cell        culture growth, wherein the instrument module and the        cultureware module are adapted for removable attachment to one        another.

In some embodiments, cultureware module includes a plurality ofbioreactors. For example, each cultureware module may have 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, or more bioreactors. In some embodiments, thebioreactors are hollow-fiber bioreactors. In embodiments in which thecultureware module has a plurality of bioreactors, the bioreactors maybe arranged in any orientation that permits production of the cellsand/or cell-derived products. For example, the bioreactors may bearranged in a line in parallel-fashion, as shown in the figures, butother orientations are possible. In some embodiments, the flow pathconnecting the bioreactors permits a first bioreactor to be inoculated(e.g., with cells and/or virus), resulting in inoculation of “downstreambioreactors” of the production module.

The production module includes two individual parts: a reusableinstrument module, and at least one disposable cell cultureware modulethat is used for a single production run and is disposable. Theinstrument module provides the hardware to support cell culture growthand production in a compact package, which is advantageous in a facilityhandling a large number of unique cell lines, for example. A pump, suchas an easy-load 4 channel peristaltic pump, moves fresh basal media intothe cultureware, removes spent media, adds high molecular weight factorand removes product harvest. A temperature controlled storage area canbe used to maintain the factor and harvest at a low temperature(preferably, approximately 4° C.). A heating mechanism can maintain thecell environment to promote growth and production. The gas blendingmechanism, in conjunction with the cultureware pH sensor controls the pHof the cell culture medium. A plurality of automated tube valving drives(e.g., two automated tube valving drives) are used to control thecultureware flow path configuration to accomplish the fluidic functionsnecessary to initiate and carryout a successful run. Valves and sensorsin the instrumentation base device control the fluid cycling in thecultureware. Drive for fluid circulation is provided. An identificationcode reader (such as a barcode reader, radio frequency identification(RFID) tag reader, bokode reader, or quick response (QR) code reader) ispreferably included to facilitate operator and lot tracing. Acommunication port preferably ties the instrument module to a facilitiesdata management system (LIMS). The instrument module of the productionmodule can include a user interface, such as a flat panel display withtouch screen, for user interaction. Likewise, an interface can beprovided for interacting with a production suite made up of a pluralityof production modules.

The one-time use cultureware is provided pre-sterilized, designed forrapid loading onto the instrument module (“quick-load”; see, forexample, WO 2007/139742 (Wojciechowski et al., “Method and System forthe Production of Cells and Cell Products and Applications Thereof”,published Dec. 6, 2007, which is incorporated herein by reference in itsentirety). The loading of the cultureware module makes connections tothe instrument module through the interface plate. Optionally, a pumpcassette, which is physically attached to the tubing, can allow the userto quickly load the pump segments. The design and layout minimizesloading errors. The cultureware enclosure provides an area that may beheated to maintain cell fluid temperature. Reservoirs to maintain fluidvolumes and cycling are included in the cultureware. Sensors for fluidcirculation rate and pH and thermal well for the instrument module'stemperature sensor are included. The blended gas from the instrumentmodule is routed to the gas exchange cartridge that provides oxygen andadds or removes carbon dioxide to the circulated fluid to support cellmetabolism. The cultureware module also includes a bioreactor (e.g.,hollow fiber bioreactor or other bioreactor type), which provides thecell space and media component exchange. Disposable containers forharvest collection and flushing can be utilized. The operator attaches amedia source, factor source, and spent media container to thecultureware before running. The media and spent media container isdisconnected, pump cassette is unloaded, cultureware body is unloadedand the used cultureware can be placed in a biohazard container fordisposal.

The present invention provides a large-scale production module forproducing cells and cell derived products in a closed, self-sufficientenvironment. More specifically, the production module allows forlarge-scale cell expansion and harvest of cells and their products withminimal need for technician interaction.

As will be described further herein, the device incorporates cellculture technology, for example, hollow fiber or similar bioreactorperfusion technology, with all tubing components, harvest tubing andtubes threaded through a pump cassette, encased in a disposablebioreactor. Following bioreactor inoculation with cells, the productionmodule follows pre-programmed processes to deliver media, maintain pH,maintain lactate levels, control temperature and harvest cells orcell-secreted protein. Standard or unique cell culture growth parameterscan be programmed such that various cell types can be expanded and suchthat virus or VLP can be harvested in an efficient, reproducible mannerwith minimal chance of human error.

The production module is based on cell growth chamber technology. Forexample, bioreactors that have a plurality of semi-permeable hollowfibers or other type of semi-permeable membrane or substrate potted(attached) in a housing to create a space inside the fiber or one sideof the membrane (referred to as intracapillary (IC) space) separate fromthat outside the fibers or on the other side of the membrane (referredto as extracapillary (EC) space). Fluid distribution between the ICspace and EC space occurs through the fiber pores which can range insize based on the application. Cells are placed on one side of the fiberor membrane, usually in the EC space, in a complete cell culture medium,which is usually the same medium used to expand cells prior tobioreactor inoculation (serum containing, serum-free, or protein-freemedium). Cells are usually placed in the EC space when secreted proteinis the desired product. In some instances, when cells are the desiredproduct, it may be beneficial to place cells in the IC space.

Medium is perfused through a bioreactor by circulating through the ICspace at a fast rate. The medium can be a liquid containing awell-defined mixture of salts, amino acids, and vitamins that oftencontain one or more protein growth factors. This serves to delivernutrients to the cell space and conversely, removes or prevents a toxicbuild-up of metabolic waste. During this circulation, medium is passedthrough an oxygenator or gas exchanger cartridge 24 which serves toprovide pH control and oxygen for the cells and conversely, removecarbon dioxide from the culture. When the bioreactor contains a smallernumber of cells, just after inoculation, the oxygenator or gas exchangecartridge is used to provide CO₂ and subsequently control pH of theculture environment. As cell number increases, the oxygenator is used toremove CO₂ which serves to enhance acid neutralization and control thepH of the culture. Other bioreactor configurations, in addition tohollow fibers, that are designed and optimized for the growth andproduction of cells and production of cell-derived products may also beused.

The production module provides significant efficiencies and costreduction through its disposable component and enclosed operation. Assuch, cell lines are contained in a closed system and continuouslycultured without the need for specialized, segregated clean rooms. Thisfully integrated apparatus eliminates the need for cleaning andsterilization validations, as well as the need for hard plumbingassociated with conventional cell culture facilities.

The production module includes two individual parts: an instrumentmodule that is reusable and an enclosed cultureware module that may beused for a single production run and is disposable. Numerous productionmodules can be used as a production suite. The instrument provides thehardware to support cell culture growth and production in a compactpackage.

An easy-load multiple channel peristaltic pump drive (see WO2007/139742, incorporated by reference) can be located in the instrumentmodule and a pump cassette can move fresh basal media into thecultureware, removes spent media, adds growth factors or othersupplements and removes product harvest. A gas exchange cartridge inconjunction with a cultureware pH sensor can be included to control thepH of the cell culture medium. Automated tube valving drives can be usedto control the cultureware flow path configuration to accomplish thefluidic switching functions needed to initiate and do a successful run.Valves and sensors can be used to control fluid cycling in thecultureware module (see WO 2007/139748 (Page et al., “Extra-CapillaryFluid Cycling System and Method for a Cell Culture Device”, publishedDec. 6, 2007, which is incorporated herein by reference in itsentirety). A pump drive for fluid circulation is provided. A wireless ortethered (attached) identification code reader (such as a barcodereader, radio frequency identification (RFID) tag reader, or quickresponse (QR) code reader) may be used by an operator to facilitate lottracing. An identification code comprises an identifier on or made partof a surface such as cultureware module or user identification tag, andwhich may include, but is not limited to, a bar code, a radio frequencyidentification tag, a number, a series of numbers, a color, a series ofcolors, a letter, a series of letters, a symbol, a series of symbols,and a combination of one or more of the foregoing. A communication portties the instrument to a data information management system (such as aMES). A user interface, such as a keyboard and/or flat panel displaywith touch screen capability is available for user interaction.

The cultureware module of the production module is providedpre-sterilized. It is designed for quick loading onto the instrumentmodule (“quick-load”), as described in WO 2007/139742 and WO 2007/139747(Page et al., “Interface of a Cultureware Module in a Cell CultureSystem and Installation Method”, published Dec. 6, 2007, which isincorporated herein by reference in its entirety). The loading of thecultureware module makes connections to the instrument. A pump cassettecan be physically attached to the tubing, allowing the user to quicklyload the pump segments. This design and layout minimizes loading errors.

The cultureware enclosure provides an area that can be heated internallyor by external devices to maintain cell fluid temperature. A fluidcycling unit can be used (see WO 2007/139748, which is incorporatedherein by reference in its entirety) to maintain fluid volumes andcycling and can be included in the production module. Sensors for fluidcirculation rate, pH and a thermal well for the instrument's temperaturesensor are provided. The blended gas from the instrument module isrouted to gas exchange cartridge that provides oxygen and adds orremoves carbon dioxide to the circulated fluid to support cellmetabolism. A magnetically coupled pump drive can be used to circulatefluid thru the bioreactor(s) and gas exchange cartridge (see, forexample, WO 2007/139742, which is incorporated by reference in itsentirety). The bioreactor that provides the cell space and mediacomponent exchange is also in the cultureware.

Cell expansion and subsequent process tracking can be facilitated bygeneration of a batch record for each culture. Historically, this isdone with a paper-based system that relies on operator input of theinformation. This is labor intensive and subject to errors. Theproduction module and production suite of the invention can incorporatean identification code reader (such as a barcode reader, radio frequencyidentification (RFID) tag reader, bokode reader, or quick response (QR)code reader), and data gathering software which, when used with theinformation management system (MES), allows for automatic generation ofthe batch record.

The production module and production suite of the present invention hasapplication in a regulated cell culture environment. It is anticipatedthat the production of viral vaccines may require the simultaneousculture of numerous cell lines in a single facility. In addition to thesegregation created through this closed culture approach, the productionmodule and production suite is designed to support a standardinformation management system (such as a LIMS or MES) protocol. Thiscapability contributes to the creation of thorough batch records andverification of culture conditions to ensure standardization, trackingand safety of each product. This capability facilitates themulti-product concept that is pivotal to facilities involved withinfectious products.

As described above, module 12 is heated to maintain cell fluidtemperature. Heating mechanism 22 (FIG. 6) maintains the cellenvironment to promote growth and production.

During installation, the cultureware module is aligned with theconnections of the instrument module and the cultureware module isplaced into the operating position as shown in FIGS. 2 and 6. All matinginterface features are functional. The cultureware module can beinserted into the instrument in an operating position with no specialoperator procedures required for loading the tubing into the clamps. Itprovides automated actuation of slide clamp, compactness, multiplelines, maintains clamp position even with loss of actuator power, lesscostly than an equivalent switching valve. Offset occluded/open positionof two tubing lines can insure a make-before-break switching of fluids.No power is required to maintain any operating position.

In one embodiment, the bioreactor is a flexible bioreactor having aflexible outer body that allows for physical movement of the cell growthsubstratum (hollow fibers, membrane or other suitable matrix) when aresultant torquing or bending moment is applied to the bioreactor ends.A flexible outer body allows for the bioreactor case to be flexed,causing fiber movement. This fiber movement enhances the release ofcells that have attached to the side of the bioreactor matrix, ifharvesting of cells is desired. The cells can then be harvested byflushing either after or during the manipulation. This method canprovide increased efficiency of cell harvest at high cell viabilitieswithout the use of chemical or enzymatic release additives.Alternatively, the bioreactor can have a rigid outer body.

Optionally, a bioreactor can be constructed using an outer housing thatincorporates a flexible center section. This center section can becomposed of a flexible, non-permeable tubing that allows each end of thebioreactor to be manipulated, thus causing movement of the growthmatrix. The purpose of this movement is to release the attachment orclumping of products on the extra-capillary (EC) side of the fibers. Theproducts can then be flushed from the EC via the access port at each endof the bioreactor.

Harvesting cells from a matrix-containing bioreactor such as a hollowfiber bioreactor has been difficult to accomplish. Typically, cells aresticky and attach themselves to the fibers or to other cells and formclusters. Rapid flushing of media through the EC space to hydraulicallyforce the cells free and into the harvest stream is the most basicmethod of harvesting cells from the EC space. Typically the quantity ofcells harvested is low because the flushing media tends to shunt throughthe EC and flush cells only from the limited fluid path.

Another method is to physically shake or impact the outer housing torelease the cells or clumps of cells. This practice may cause physicaldamage to the bioreactor or its associated components. Another methodincludes the use of chemicals to disrupt the adhesion of cells to thefibers or to disrupt the clumps of cells. Adding chemicals to acontrolled process may cause adverse effects on cell viability and canintroduce an unwanted agent in the down-stream processing.

The production module and production suite of the invention fullyintegrates the concept of disposable cultureware into automated processcontrol for maintaining and expanding specialized cells (primary cellsor cell lines) for a duration of any time needed. To accomplish this,the apparatus of the present invention was designed for EC space fluidflow that enhances cell growth in high density perfusion culture, yetremains completely closed and disposable. The integrated pre-assembledcultureware, which includes all tubing, bioreactor, oxygenator, and pHprobe, is enclosed in a single unit that easily snaps into theapparatus. In addition to this error-proof, quick-load design, theentire cultureware unit enclosed by the casing becomes the cell cultureincubator with temperature control regulated through automated processcontrol of the instrument. Pumps and fluid control valves facilitatedisposability and error-proof installation, eliminating the possibilityof technician mistakes. Finally, during the course of any culture, theclosed system can have restricted access except for trained andauthorized personnel. For example, manipulations or sampling, outside ofprogram parameters, can require password and identification code (e.g.,bar code) access before they can be implemented.

II. PRODUCTION SUITE

Another aspect of the invention concerns a production suite comprising aplurality of production modules of the invention, functionallyconnected. In some embodiments, the flow path connecting the pluralityof production modules permits a first production module to be inoculated(e.g., with cells and/or virus), resulting in inoculation of “downstreamproduction modules”. Virtually any number of production molecules perproduction suite is possible.

The production suite may further include a room for enclosing theplurality of production modules and having one or more support surfacesfor supporting the plurality of production modules. Preferably, the roomis environment-controlled (e.g., temperature-controlled) to achieve thedesired cell culture conditions. Consequently, no further heat/coolingsource is required locally at the site of the bioreactor(s). In someembodiments, the room is a modular and/or relocatable building. Forexample, the building may have one or more receivers affixed to thebuilding frame or wall, for receiving a lifting attachment allowingtransport of the building onto a truck, trailer, vessel, aircraft, orother conveyance. The building may include one or more anchors foranchoring the building to the ground.

Another aspect of the invention is a method for large-scale productionof cells and/or cell-derived products, comprising providing one or moreproduction modules or one or more production suites of any precedingclaim, introducing cells into the one or more bioreactors; culturing thecells to produce grown cells and/or cell-derived products; andharvesting the grown cells and/or cell-derived products. Any desiredcell type may be used, e.g., mammalian cells, insect cells, avian cells,or plant cells. Any desired cell-derived product may be produced if asatisfactory cell type(s) is available, such as immunoglobulins,proteins, viruses, and virus-like particles.

Medium is perfused through one or more of the bioreactors of thecultureware module. The medium can be a liquid containing a well-definedmixture of salts, amino acids, and vitamins that often contain one ormore protein growth factors. This serves to deliver nutrients to thecell space and conversely, removes or prevents a toxic build-up ofmetabolic waste. During this circulation, medium is passed through anoxygenator or gas exchanger cartridge (FIGS. 3 and 5) which serves toprovide pH control and oxygen for the cells and conversely, removecarbon dioxide from the culture. When the bioreactor contains a smallernumber of cells, just after inoculation, the oxygenator or gas exchangecartridge can be used to provide CO₂ and subsequently control pH of theculture environment. As cell number increases, the oxygenator is used toremove CO₂ which serves to enhance acid neutralization and control thepH of the culture.

The following is an example of a cell culture run. An operator removesthe sterile cultureware from its packaging and mounts it to theinstrument module. Mechanical interfacing occurs automatically when thecultureware is inserted. Cultureware module information is scanned bythe instrument and stored for the batch record. Sterile connections aremade to the media source, waste factor and harvest (FIG. 5) vessels. Theoperator starts the process and the instrument automatically sequencesthrough the process of flushing and preparing the cultureware module forinoculation. Throughout the run, online intercapillary metabolicanalysis (FIG. 5) can be used to verify the sensor array and provideadditional information to the instrument. Just before inoculation,factor supplemented media is introduced into the extracapillary space.Once prepared, the desired cells can be introduced locally through theInoculum-In (pumped into the bioreactors) or diverted from neighboringcultureware modules (FIGS. 4 and 5). The instrument sequence shifts intoa growth mode to expand the cell mass. Medium is perfused through thebioreactors of the cultureware module. This serves to deliver nutrientsto the cell space and conversely, removes or prevents a toxic build-upof metabolic waste. During this circulation, medium is passed through anoxygenator or gas exchanger cartridge (FIGS. 3 and 5) which serves toprovide pH control and oxygen for the cells and conversely, removecarbon dioxide from the culture. When the bioreactor contains a smallernumber of cells (e.g., immediately after inoculation), the oxygenator orgas exchange cartridge can be used to provide CO₂ and subsequentlycontrol pH of the culture environment using the sensors built into thecultureware module (FIG. 5). As cell number increases, the oxygenator isused to remove CO₂ which serves to enhance acid neutralization andcontrol the pH of the culture. The online extracapillary biochemicalanalysis (FIG. 5) provides information concerning the productconcentration or cell mass. In the case of virus production, when thecell mass is determined to be optimal (for example, after a number ofdays), the operator will initiate introduction of virus and/orvirus-infected cells (e.g., Influenza virus and/or Influenza infectedcells) into the cell mass locally through the Inoculum-In (pumped intothe bioreactors) or diverted from neighboring infected culturewaremodules (FIGS. 4 and 5). After an infection period, harvesting of theinfectious (plaque-forming) virus is started, which can be locally fromthe harvest line or network-wide from the Inoculum-Out line.Alternatively, if the product is not a virus (e.g., cells orbiomolecules such as antibodies or other polypeptides), agents such ascytokines can be introduced to the cells to induce a desired response orproduction of a desired product. When a significant drop in one or moremetabolics is observed, harvesting is stopped. The operator removes theharvest, if local, and indicates the run is complete. The instrumentmodule prepares for cultureware removal. Sterile disconnects are madefor the inoculum, media and waste lines. The cultureware module isremoved and preferably disposed of as a biological hazard. Theinstrument module surfaces can be spray cleaned. The instrument is thenready for the next production run.

A wide variety of media, salts, media supplements, and products formedia formulation can be utilized to produce the cells and cell-derivedproducts, depending upon the particular cell type or types. Examples ofthese substances include, but are not limited to, carrier and transportproteins (e.g., albumin), biological detergents (e.g., to protect cellsfrom shear forces and mechanical injury), biological buffers, growthfactors, hormones, hydrosylates, lipids (e.g., cholesterol), lipidcarriers, essential and non-essential amino acids, vitamins, sera (e.g.,bovine, equine, human, chicken, goat, porcine, rabbit, sheep), serumreplacements, antibiotics, antimycotics, and attachment factors. Thesesubstances can be present in various classic and/or commerciallyavailable media, which can also be utilized with the subject invention.Examples of such media include, but are not limited to, Ames' Medium,Basal Medium Eagle (BME), Click's Medium, Dulbecco's Modified Eagle'sMedium (DMEM), DMEM/Nutrient Mixture F12 Ham, Fischer's Medium, MinimumEssential Medium Eagle (MEM), Nutrient Mixtures (Ham's), WaymouthMedium, and William's Medium E.

The cell culture environment within each bioreactor can be manipulatedindividually, or as a group, by exposing the cells to agents/techniques(e.g., cytokines, hormones, feed strategies, temperatures, growth orcell cycle inhibitors, co-cultures) to evoke a desirable response fromthe process. This can, for example, enhance productivity, shortenprocess times and/or produce secondary effects or products. One or moreof such agents can be included as a component of media or added to theculture environment. For example, stem cells can be exposed to agents toinduce or inhibit differentiation, and lymphocytes can be exposed tospecific antigens to produce a T-cell response.

III. CELL PRODUCTION

The subject invention provides a ready source of cells for medicine andresearch, including pharmacological studies for the screening of variousagents, and toxicologic studies for the cosmetic and pharmaceuticalindustries. Any cells desired for cell production, or any cells usefulfor production of the cell-derived product of interest can be used inthe production module, production suite, and methods of the invention.For example, cells may be human or non-human mammalian cells, insectcells, avian cells, or plant cells. The cells may be transformed ornon-transformed cell lines, primary cells including somatic cells suchas lymphocytes or other immune cells, chondrocytes, myocytes ormyoblasts, epithelial cells and patient specific cells, primary orotherwise. Included also are cells or cell lines that have beengenetically modified, such as non-stem cells, adult stem cells andembryonic stem cells.

The various methods employed in the genetic modification of cells arewell known in the art and are described, for example, in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, second edition, volumes 13, Cold Spring Harbor Laboratory, New York, and Gloves, D. M. (1985) DNACloning, Vol. I: A Practical Approach, IRL Press, Oxford. Thus, it iswithin the skill of those in the genetic engineering art to extract DNAfrom its source, perform restriction enzyme digestions, electrophoreseDNA fragments, tail and anneal plasmid and insert DNA, ligate DNA,transform cells, e.g., prokaryotic and eukaryotic cells, prepare plasmidDNA, electrophorese proteins, and sequence DNA.

The cells grown using the invention can range in plasticity fromtotipotent or pluripotent stem cells (e.g., adult or embryonic),precursor or progenitor cells, to highly specialized cells, such asthose of the central nervous system (e.g., neurons and glia). In someembodiments, the cells are bone marrow cells, hematopoietic stem cellsor hematopoietic progenitor cells, mesenchymal stem cells, or other stemcells or progenitor cells. The cells may be administered to a subject inan enriched (e.g., purified or isolated) or non-enriched form. Stem andprogenitor cells can be obtained from a variety of sources, includingembryonic tissue, fetal tissue, adult tissue, umbilical cord blood,peripheral blood, bone marrow, and brain, for example.

In some embodiments, the cells are human cells. However, it will beunderstood by one of skill in the art that the present invention is alsoapplicable for veterinary purposes. Cells of non-human animals can findapplication either in human or animal subjects (transplant recipients).For example, although dopamine neurons from human, pig, and rat aresimilar in that they synthesize dopamine and release synaptically intothe brain, they differ immunologically, in extent of reinervation of thebrain, in life span, and in infection agents associated with thespecific donor or donor species. These traits can be exploited for theirspecific strengths and weaknesses.

As will be understood by those skilled in the art, there are over 200cell types in the human body. The production modules, production suites,and methods of the invention can be used to grow any of these celltypes. For example, cells can include those cells arising from theectoderm, mesoderm, or endoderm germ cell layers. Such cells include,but are not limited to, bone marrow cells, neurons, glial cells(astrocytes and oligodendrocytes), muscle cells (e.g., cardiac,skeletal), chondrocytes, fibroblasts, melanocytes, Langerhans cells,keratinocytes, endothelial cells, epithelial cells, pigment cells (e.g.,melanocytes, retinal pigment epithelial (RPE) cells, iris pigmentepithelial (IPE) cells), hepatocytes, microvascular cells, pericytes(Rouget cells), blood cells (e.g., erythrocytes), cells of the immunesystem (e.g., B and T lymphocytes, plasma cells, macrophages/monocytes,dendritic cells, neutrophils, eosinophils, mast cells), thyroid cells,parathyroid cells, pituitary cells, pancreatic cells (e.g.,insulin-producing beta cells, glucagon-producing alpha cells,somatostatin-producing delta cells, pancreatic polypeptide-producingcells, pancreatic ductal cells), stromal cells, adipocytes, reticularcells, rod cells, and hair cells. Other examples of cell types that canbe grown include those disclosed by Spier R. E. et al., eds., (2000) TheEncyclopedia of Cell Technology, John Wiley & Sons, Inc., and Alberts B.et al., eds., (1994) Molecular Biology of the Cell, 3^(rd) ed., GarlandPublishing, Inc., e.g., pages 1188-1189.

Various cell lines have also been used for a variety of purposes, andcan be grown using the production modules, production suites, andmethods of the invention. Fetal kidney cells and amniotic cells havebeen transplanted as sources of trophic factors. Adrenal medullarycells, sympathetic ganglion cells, and carotid body cells have beentransplanted as sources of dopamine. Fibroblasts and glial cells havebeen transplanted as sources of trophic factors, to carry genes throughrecombinant strategies, or for demyelinating diseases, for example.Corneal endothelial cells have been used for corneal transplants.Myoblasts have been transplanted for the treatment of muscular dystrophyand cardiac disease. Other cell lines include pancreatic islet cells fordiabetes; thyroid cells for thyroid disorders; blood cells for AIDS,bone marrow transplant, and inherited disorders; bone and cartilage forosteoarthritis, rheumatoid arthritis, or for fracture repair; skin orfat cells for reconstructive purposes, such as in skin grafts afterburns or cosmetic surgery; breast augmentation with fat; hair folliclereplacement; liver cells for liver disorders inducing hepatitis; andretinal pigment epithelial cells (RPE) for retinitis pigmentosa andParkinson's disease.

The cells to be used in the various aspects of the present invention arepreferably mammalian cells. They may be of human or animal origin.Examples of mammalian cells that can be grown using the productionmodules, production suites, and methods of the invention include, butare not limited to, murine C127 cells, 3T3 cells, COS cells, humanosteosarcoma cells, MRC-5 cells, BHK cells, VERO cells, CHO (Chinesehamster ovary) cells, HEK 293 cells, rHEK 293 cells, normal humanfibroblast cells, Stroma cells, Hepatocytes, or PER.C6 cells. Examplesof hybridomas that may be cultured in the process according to thepresent invention include, e.g., DA4.4 cells, 123A cells, 127A cells,GAMMA cells and 67-9-B cells.

Stem cells are believed to have immense potential for therapeuticpurposes for numerous diseases. Stem cells have been derived fromnumerous donor sources, including, but not limited to, embryonic, blast,tissue-derived, blood, and cord-blood cells; organ-derived progenitorcells; and bone marrow stromal cells, among others. Such stem cells canbe differentiated along numerous pathways to produce virtually any celltype. These cells can be transplanted either before or afterdifferentiation. Hematopoietic stem cells (HSC) have been used for manyyears, and typically used for treatment of hematopoietic cancers (e.g.,leukemias and lymphomas), non-hematopoietic malignancies (cancers inother organs). Other indications include diseases that involve geneticor acquired bone marrow failure, such as aplastic anemia, thalassemiasickle cell anemia, and autoimmune diseases.

Methods and markers commonly used to identify stem cells and tocharacterize differentiated cell types are described in the scientificliterature (e.g., Stem Cells: Scientific Progress and Future ResearchDirections, Appendix E1-E5, report prepared by the National Institutesof Health, June, 2001). The list of adult tissues reported to containstem cells is growing and includes bone marrow, peripheral blood,umbilical cord blood, brain, spinal cord, dental pulp, blood vessels,skeletal muscle, epithelia of the skin and digestive system, cornea,retina, liver, and pancreas.

Optionally, stem cells may be induced to differentiate using varioustechniques or exposure to agents (differentiation-inducing agents). Forexample, depending upon the cell type, stem cells may induced todifferentiate along certain lineages in cell culture by applyingmechanical force (e.g., compressive forces or pressure, tensile forces(e.g., mechanical loading or stretch), and fluid-applied forces (shearflow)), or by contacting the cells with one or moredifferentiation-inducing agents (e.g., trophic factors, hormonalsupplements), such as forskolin, retinoic acid, putrescin-transferrin,cholera toxin, insulin-like growth factor (IGF), transforming growthfactor (e.g., TGF-.alpha., TGF-.beta.), tumor necrosis factor (TNF),fibroblast growth factor (FGF), epidermal growth factor (EGF),granulocyte macrophage-colony stimulating factor (GM-CSF), hepatocytegrowth factor (HGF), hedgehog, vascular endothelial growth factor(VEGF), thyrotropin releasing hormone (TRH), platelet derived growthfactor (PDGF), sodium butyrate, butyric acid, cyclic adenosinemonophosphate (cAMP), cAMP derivatives (e.g., dibutyryl cAMP,8-bromo-cAMP) phosphodiesterase inhibitors, adenylate cyclaseactivators, prostaglandins, ciliary neurotrophic factor (CNTF),brain-derived neurotrophic factor (BDNF), neurotrophin 3, neurotrophin4, interleukins (e.g., IL-4), interferons (e.g., interferon-gamma),leukemia inhibitory factor (LIF), potassium, amphiregulin, dexamethasone(glucocorticoid hormone), isobutyl 3-methyulxanthine, somatostatin,lithium, and growth hormone.

Cells and cells-derived products can be harvested using methods known inthe art. Various biomolecules produced by genetically modified ornon-genetically modified cells that are produced using the productionmodules, production suites, and methods of the invention can beharvested (e.g., isolated from the biomolecule-producing cells) forvarious uses, such as the production of drugs and for pharmacologicalstudies. Thus, using the production modules, production suites, andmethods of the invention, cells can be used as biological “factories” toprovide the product of exogenous DNA and/or the natural product of thecells in vitro, or in vivo within an animal. The term “biomolecule”refers to a molecule or molecules that can be produced by cells (acell-derived product). Such biomolecules include, but are not limitedto, proteins, peptides, amino acids, lipids, carbohydrates, nucleicacids, nucleotides, viruses, and other substances. Some specificexamples of biomolecules include trophic factors, hormones, and growthfactors, such as brain-derived growth factor (BDNF) and glial-derivedneurotrophic factor (GDNF). For example, pituitary cells can be grown toproduce growth hormone; kidney cells can be grown to produce plasminogenactivator; bone cells can be grown to produce bone morphogenetic protein(BMP) or other proteins involved in bony fusions or prosthetic surgery.Hepatitis-A antigen can be produced from liver cells. Cells can be grownto produce various viral vaccines and antibodies. Interferon, insulin,angiogenic factor, fibronectin and numerous other biomolecules can beproduced by growing cells and harvesting these products. Thebiomolecules can be intracellular, transmembrane, or secreted by thecells, for example.

Cells produced using the invention can be administered to humans oranimals as cell therapy to alleviate the symptoms of a wide variety ofdisease states and pathological conditions, in various stages ofpathological development. For example, cells can be used to treat acutedisorders (e.g., stroke or myocardial infarction), and administeredacutely, subacutely, or in the chronic state. Similarly, the cells ofthe subject invention can be used to treat chronic disorders (e.g.,Parkinson's disease, diabetes, or muscular dystrophy), and administeredpreventatively and/or prophylactically, early in the disease state, inmoderate disease states, or in severe disease states. For example, thecells can be administered to a target site or sites on or within apatient in order to replace or compensate for the patient's own damaged,lost, or otherwise dysfunctional cells. This includes infusion of thecells into the patient's bloodstream. The cells to be administered canbe cells of the same cell type as those damaged, lost, or otherwisedysfunctional, or a different cell type. As used herein, patients “inneed” of the cells of the subject invention include those desiringelective surgery, such as elective cosmetic surgery.

The cells of the invention can be administered as autografts, syngeneicgrafts, allografts, and xenografts, for example. As used herein, theterm “graft” refers to one or more cells intended for implantationwithin a human or non-human animal. Hence, the graft can be a cellularor tissue graft, for example. Cells can be administered to a patient byany method of delivery, such as intravascularly, intracranially,intracerebrally, intramuscularly, intradermally, intravenously,intraocularly, orally, nasally, topically, or by open surgicalprocedure, depending upon the anatomical site or sites to which thecells are to be delivered. The cells can be administered to a patient inisolation or within a pharmaceutical composition comprising the cellsand a pharmaceutically acceptable carrier. As used herein, apharmaceutically acceptable carrier includes solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic agents, and thelike. Pharmaceutical compositions can be formulated according to knownmethods for preparing pharmaceutically useful compositions. Formulationsare described in a number of sources that are well known and readilyavailable to those of ordinary skill in the art. For example,Remington's Pharmaceutical Science (Martin E. W., Easton Pa., MackPublishing Company, 19th ed.) describes formulations that can be used inconnection with the subject invention. Formulations suitable forparenteral administration, for example, include aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions that may include suspending agents and thickening agents. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the subject invention can includeother agents conventional in the art having regard to the type offormulation and route of administration in question.

IV. POLYPEPTIDE PRODUCTION

The biomolecule can be a polypeptide of interest, such as a naturallysecreted protein, a normally cytoplasmic protein, a normallytransmembrane protein, or a human or a humanized antibody. When theprotein of interest is a naturally cytoplasmic or a naturallytransmembrane protein, the protein has preferably been engineered inorder to become soluble and secreted, i.e., by placing a signal peptidein front of it or of a (soluble or extracellular) fragment of it.However, intracellular biomolecules may also be harvested by lysing thecells.

The polypeptide of interest may be of any origin. Some polypeptides ofinterest are of human origin, and may be therapeutic proteins. Forexample, the protein of interest may be selected from a hormone, acytokine-binding protein, an interferon, a soluble receptor, or anantibody. Therapeutic proteins that may be produced include, forexample, chorionic gonadotropin, follicle-stimulating hormone,lutropin-choriogonadotropic hormone, thyroid stimulating hormone, growthhormone, in particular human growth hormone, interferons (e.g.,interferon beta-1a, interferon beta-1b), interferon receptors (e.g.,interferon gamma receptor), TNF receptors p55 and p75, and solubleversions thereof, TACI receptor and Fc fusion proteins thereof,interleukins (e.g., interleukin-2, interleukin-11), interleukin bindingproteins (e.g., interleukin-18 binding protein), anti-CD11a antibodies,erythropoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony-stimulating factor, pituitary peptidehormones, menopausal gonadotropin, insulin-like growth factors (e.g.,somatomedin-C), keratinocyte growth factor, glial cell line-derivedneurotrophic factor, thrombomodulin, basic fibroblast growth factor,insulin, Factor VIII, somatropin, bone morphogenetic protein-2,platelet-derived growth factor, hirudin, epoietin, recombinantLFA-3/IgG1 fusion protein, glucocerebrosidase, and muteins, fragments,soluble forms, functional derivatives, fusion proteins thereof. In someembodiments, the polypeptide is selected from the group consisting ofchorionic gonadotropin (CG), follicle-stimulating hormone (FSH),lutropin-choriogonadotropic hormone (LH), thyroid stimulating hormone(TSH), human growth hormone (hGH), interferons (e.g., interferonbeta-1a, interferon beta-1b), interferon receptors (e.g., interferongamma receptor), TNF receptors p55 and p75, interleukins (e.g.,interleukin-2, interleukin-11), interleukin binding proteins (e.g.,interleukin-18 binding protein), anti-CD11a antibodies, and muteins,fragments, soluble forms, functional derivatives, fusion proteinsthereof.

Further preferred polypeptides of interest include, e.g.,erythropoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony-stimulating factor, pituitary peptidehormones, menopausal gonadotropin, insulin-like growth factors (e.g.,somatomedin-C), keratinocyte growth factor, glial cell line-derivedneurotrophic factor, thrombomodulin, basic fibroblast growth factor,insulin, Factor VIII, somatropin, bone morphogenetic protein-2,platelet-derived growth factor, hirudin, epoietin, recombinantLFA-3/IgG1 fusion protein, glucocerebrosidase, and muteins, fragments,soluble forms, functional derivatives, fusion proteins thereof.

Proteins that can be produced by the present invention include tumorantigens and antibodies. An epitope of the tumor antigen can be any siteon the antigen that is reactive with an antibody or T cell receptor.Other examples of tumor antigens include, but are not limited to humanepithelial cell mucin (Muc-1; a 20 amino acid core repeat for Muc-1glycoprotein, present on breast cancer cells and pancreatic cancercells), the Ha-ras oncogene product, p53, carcino-embryonic antigen(CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3,tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 & 2,HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP),CO17-1A, GA733, gp72, p53, the ras oncogene product, HPV E7 and melanomagangliosides, as well as any other tumor antigens now known oridentified in the future. In some embodiments, the tumor antigen is theidiotype of a B-cell derived lymphoma (e.g., IgM or IgG isotype).Various antibody isotypes may be produced using the invention, includingIgG, IgM, IgA, IgD, and IgE. In some embodiments, the product is anantibody, which may then be conjugated to a carrier molecule, such as acarrier protein (e.g., keyhole limpet hemocyanin (KLH)).

The subject invention provides a ready source of polypeptides formedicine and research. Polypeptides produced using the invention can beadministered to a human or non-human animal by any method of delivery,such as intravascularly, intracranially, intracerebrally,intramuscularly, intradermally, intravenously, intraocularly, orally,nasally, topically, or by open surgical procedure, depending upon theanatomical site or sites to which the polypeptides are to be delivered.The polypeptides can be administered to a patient in isolation or withina pharmaceutical composition comprising the polypeptide and apharmaceutically acceptable carrier. As used herein, a pharmaceuticallyacceptable carrier includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, and the like.Pharmaceutical compositions can be formulated according to known methodsfor preparing pharmaceutically useful compositions. Formulations aredescribed in a number of sources that are well known and readilyavailable to those of ordinary skill in the art. For example,Remington's Pharmaceutical Science (Martin E. W., Easton Pa., MackPublishing Company, 19th ed.) describes formulations that can be used inconnection with the subject invention. Formulations suitable forparenteral administration, for example, include aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions that may include suspending agents and thickening agents. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the subject invention can includeother agents conventional in the art having regard to the type offormulation and route of administration in question.

V. VIRUS AND VIRUS-LIKE PARTICLE PRODUCTION

Viruses, virus-like particles (VLP), and viral vectors represent anothertype of cell-derived product that may be produced using the productionmodules, production suites, and methods of the invention. Viruses, VLPs,and viral vectors can be produced with the invention using cell typesutilized for propagating the virus of interest. Examples of mammaliancells useful for production of virus include Madin-Darby canine kidney(MDCK) cells, VERO cells, or other monolayer cell types. The cells aregrown in the bioreactor of the invention and, after a sufficient cellnumber is reached, can then be infected with the virus or viral vector,which spreads throughout the culture and larger quantities of virus orvector is then harvested. Alternatively, cells can be infected prior toinoculation of the bioreactor with the cells. The harvested virus andvectors can be used, for example, for vaccines and/or as gene deliveryvectors. For example, influenza virus can be grown and vaccines forinfluenza produced from the harvested virus. While the roller-bottle andegg-based vaccine production processes remain relatively reliable, anefficient cell-based production system would represent a significantimprovement in providing a faster, less-expensive, and less cumbersomemethod of growing viruses.

Madin-Darby canine kidney (MDCK) cells and African green monkey kidney(Vero) cells are useful for the production of influenza virus. Othercontinuous cell lines suitable as host cells for production of humanvaccines include human fetal retina (Per.C6) and duck embryonic retina(AGE1.CR) cells. In addition to production of virus, the invention maybe used for growth and expansion of virus-infected cells, and cellculture-based production of viruses products, e.g., production of virus,viral vaccines, viral proteins, viral vectors for gene delivery, andVLPs.

In some embodiments, the virus is influenza A, B or C. In someembodiments, the virus is influenza A strain H5N1 or H1N1. In someembodiments, the virus is an orthomyxovirus, paramyxovirus, arbovirus,filovirus, enterovirus, rhinovirus, herpes virus, or hepadina virus.

The methods for production of virus (including viral vectors) and VLPsinvolving culturing the virus-infected cells under conditions that allowfor regulation of the concentration of a molecule inhibitory to virus orVLP yield (such as non-structural (NS) protein, viral ribnucleoprotein(RNP), etc.), as described in WO 2012/171026 (Hirschel et al., “Methodsfor High Yield Virus Production”, published Dec. 13, 2012), which isincorporated herein by reference in its entirety, may be utilized withthis invention. In an embodiment, these methods for the production ofvirus and VLP comprise culturing virus-infected cells in a bioreactorcomprising a first compartment, a second compartment, and a membraneseparating the first and second compartments, wherein the cells arecultured in the first compartment under conditions that allow forregulating the concentration of a molecule inhibitory to virus or VLPyield from the cells within the first compartment, and allow forproduction of the virus or VLP at a yield greater than that achieved inthe absence of said regulation. In another embodiment, the methodinvolves regulating the concentration of a molecule inhibitory to viralyield or VLP yield in a first compartment of a bioreactor comprising thefirst compartment, a second compartment, and a membrane separating thefirst and second compartments, the method comprising the culturingvirus-infected cells in the first compartment by adding cell culturemedium to the first compartment and controlling how rapidly culturemedium bearing the inhibitor molecule is replaced with fresh culturemedium in the first compartment, or by diffusing one or more inhibitormolecules from the first compartment, through the membrane, into thesecond compartment.

The following is an example of a cell culture run for virus production.An operator removes the sterile cultureware from its packaging andmounts it to the instrument module. Mechanical interfacing occursautomatically when the cultureware is inserted. Cultureware moduleinformation is scanned by the instrument and stored for the batchrecord. Sterile connections are made to the defined media source, wastefactor and harvest (FIG. 5) vessels. The operator starts the process andthe instrument automatically sequences through the process of flushingand preparing the cultureware module for inoculation. Throughout therun, online intercapillary metabolic analysis (FIG. 5) can be used toverify the sensor array and provide additional information to theinstrument. Just before inoculation, factor supplemented media isintroduced into the extracapillary space. Once prepared, MDCK(Madin-Darby Canine Kidney) cells or other host cells appropriate forproduction of the virus can be introduced locally through theInoculum-In (pumped into the bioreactors) or diverted from neighboringcultureware modules (FIGS. 4 and 5). The instrument sequence shifts intoa growth mode to expand the cell mass. Medium is perfused through thebioreactors of the cultureware module. This serves to deliver nutrientsto the cell space and conversely, removes or prevents a toxic build-upof metabolic waste. During this circulation, medium is passed through anoxygenator or gas exchanger cartridge (FIGS. 3 and 5) which serves toprovide control and oxygen for the cells and conversely, remove carbondioxide from the culture. When the bioreactor contains a smaller numberof cells (e.g., immediately after inoculation), the oxygenator or gasexchange cartridge can be used to provide CO₂ and subsequently controlpH of the culture environment using the sensors built into thecultureware module (FIG. 5). As cell number increases, the oxygenator isused to remove CO₂ which serves to enhance acid neutralization andcontrol the pH of the culture. The online extracapillary biochemicalanalysis (FIG. 5) provides information concerning the productconcentration or cell mass. After a number of days when the cell mass isdetermined to be optimal, the operator will initiate introduction ofvirus and/or virus-infected cells (e.g., Influenza virus and/orInfluenza infected cells) into the cell mass locally through theInoculum-In (pumped into the bioreactors) or diverted from neighboringinfected cultureware modules (FIGS. 4 and 5). After an infection period,harvesting of the infectious (plaque-forming) virus is started, whichcan be locally from the harvest line or network-wide from theInoculum-Out line. When a significant drop in one or more metabolics isobserved, harvesting is stopped. The operator removes the harvest, iflocal, and indicates the run is complete. The instrument module preparesfor cultureware removal. Sterile disconnects are made for the inoculum,media and waste lines. The cultureware module is removed and preferablydisposed of as a biological hazard. The instrument module surfaces canbe spray cleaned. The instrument is then ready for the next productionrun.

The process of manufacturing a viral vaccine comprises the process ofreplicating a virus using a production module, production suite, and/ormethod of the invention and harvesting the virus or VLP, which caninclude at least one step selected among filtering, concentrating,freezing and stabilizing by addition of a stabilizing agent. The virusharvest can be performed according to technologies well-known to the manskilled in the art. According to a preferred embodiment, the step ofharvesting the virus comprises collecting cell culture supernatantobtained from centrifugation, then filtering, concentrating, freezingand stabilizing virus preparation by addition of stabilizing agent. Forexample, for influenza virus, see Furminger, In Nicholson, Webster andHay (Eds) Textbook of influenza, chapter 24 pp 324-332.

The process of manufacturing a viral vaccine according to the inventionmay also comprise the additional step of inactivation of harvestedvirus. Inactivation can be performed by treatment with formaldehyde,beta-propiolactone, ether, ether and detergent (i.e., such as Tween80™), cetyl-trimethyl ammonium bromide (CTAB) and Triton N102, sodiumdeoxycholate and tri(N-butyl)phosphate.

The production module, production suite, and methods of the inventionmay also be used for preparation of viral antigenic proteins from thevirus produced therewith. The method further comprises the additionalsteps of: a) optionally, incubating cell culture supernatant comprisingwhole virus harvested from the bioreactor with a desoxyribonucleic acidrestriction enzyme, preferably DNAses and nucleases (preferably, the DNAdigestion enzyme is benzonase (Benzon nuclease) or DNase I); b)adjunction of cationic detergent (examples of cationic detergent are;without limitation: cetyl-trimethyl ammonium salt such as CTAB,myristyl-trimethyl ammonium salt, lipofectine, DOTMA and Tween™); c)isolation of antigenic proteins. This later step may be carried out bycentrifugation or ultrafiltration.

The virus in the vaccine may be present either as intact virusparticles, or as disintegrated virus particles. According to anembodiment, the vaccine is a killed or inactivated vaccine. According toanother embodiment, the vaccine is a live attenuated vaccine. Accordingto a third embodiment, the vaccine comprises viral antigenic proteinsobtainable from a virus prepared according to the method of theinvention.

The vaccine may comprise the virus in combination with pharmaceuticallyacceptable substances which increase the immune response. Non-limitingexamples of substances which increase the immune response comprisesincomplete Freund adjuvant, saponine, aluminium hydroxide salts,lysolecithin, plutonic polyols, polyanions, peptides, bacilliCalmette-Guerin (BCG) and corynebacterium parvum. In addition,immuno-stimulating proteins (e.g., interleukins IL-1, IL-2, IL-3, IL-4,IL-12, IL-13, granulocyte-macrophage-colony-stimulating factor) may beused to enhance the vaccine immune response.

The vaccine may be a liquid formulation, a frozen preparation, adehydrated and frozen preparation, or adapted to intra-nasal route ofadministration, for example.

The vaccine may be used for the prophylactic and/or therapeutictreatment of a human infected by a virus or at risk of infection, or fortreatment or prevention of other diseases such as cancer. The viralvaccine may be a recombinant viral vaccine.

The subject invention provides a ready source of vaccines for medicineand research. Vaccines produced using the invention can be administeredto a human or non-human animal by any method of delivery, such asintravascularly, intracranially, intracerebrally, intramuscularly,intradermally, intravenously, intraocularly, orally, nasally, ortopically, depending upon the anatomical site or sites to which thevaccines are to be delivered. The vaccines can be administered to apatient in isolation or within a pharmaceutical composition comprisingthe vaccine and a pharmaceutically acceptable carrier. As used herein, apharmaceutically acceptable carrier includes solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic agents, and thelike. Pharmaceutical compositions can be formulated according to knownmethods for preparing pharmaceutically useful compositions. Formulationsare described in a number of sources that are well known and readilyavailable to those of ordinary skill in the art. For example,Remington's Pharmaceutical Science (Martin E. W., Easton Pa., MackPublishing Company, 19th ed.) describes formulations that can be used inconnection with the subject invention. Formulations suitable forparenteral administration, for example, include aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient; and aqueous and nonaqueous sterilesuspensions that may include suspending agents and thickening agents. Itshould be understood that in addition to the ingredients particularlymentioned above, the formulations of the subject invention can includeother agents conventional in the art having regard to the type offormulation and route of administration in question.

VI. COMPUTER IMPLEMENTATION AND COMPUTER READABLE MEDIA

Aspects of the invention can be described in the general context ofcomputer-executable instructions, such as program modules, beingexecuted by a computer. Generally, program modules include routines,programs, objects, components, data structures, etc., that performparticular tasks or implement particular abstract data types. Suchprogram modules can be implemented with hardware components, softwarecomponents, or a combination thereof. Moreover, those skilled in the artwill appreciate that the invention can be practiced with a variety ofcomputer-system configurations, including multiprocessor systems,microprocessor-based or programmable-consumer electronics,minicomputers, mainframe computers, and the like. Any number ofcomputer-systems and computer networks are acceptable for use with thepresent invention.

Specific hardware devices, programming languages, components, processes,protocols, formats, and numerous other details including operatingenvironments and the like are set forth to provide a thoroughunderstanding of the present invention. In other instances, structures,devices, and processes are shown in block-diagram form, rather than indetail, to avoid obscuring the present invention. But anordinary-skilled artisan would understand that the present invention canbe practiced without these specific details. Computer systems, servers,work stations, and other machines can be connected to one another acrossa communication medium including, for example, a network or networks.

As one skilled in the art will appreciate, embodiments of the presentinvention can be embodied as, among other things: a method, system, orcomputer-program product. Accordingly, the embodiments can take the formof a hardware embodiment, a software embodiment, or an embodimentcombining software and hardware. In an embodiment, the present inventiontakes the form of a computer-program product that includescomputer-useable instructions embodied on one or more computer-readablemedia. Methods, data structures, interfaces, and other aspects of theinvention described above can be embodied in such a computer-programproduct.

Software can provide for sequenced operation of an individual productionmodule or a group of functionally connected production modules (aproduction suite). System security (operator identification, passwordprotection, multi-tiered users, alarm notification and operational togfor batch record) can be provided. Transactional interaction with aManufacturing Execution System (MES) is supported. In one embodiment,the software provides sequenced operation that includes one or more (andpreferably all) of the following modes or phases comprising thedescribed steps or operation status:

-   -   Idle—non-running state. Optionally, during the idle mode, access        to historical data, calibration and test.    -   Load—Mounting of the cultureware module onto the instrument        module.    -   Acceptance Testing—checking the integrity of the loaded        cultureware module,    -   Flush—Filling fluid and exchanging additional volume to purge        wetting components from the bioreactors.    -   Pre-inoculation—circulating fluid and stabilizing cultureware        operation. Optionally, range testing of sensing elements can be        performed during the pre-inoculation mode. Factors can be        introduced at the end of this step.    -   Inoculation—Moving inoculum cells and/or virus) from source        (e.g., from a local container or networked from another        production module).    -   Growth—Modifying the cell culture environment by        manipulating/maintaining pH, circulation flow, media feed,        factor addition, EC fluid removal and EC fluid cycling to        optimize cell mass increase or productivity. This can be based        on sensor readings (on-line and off-line), a pre-determined        sequence or a combination of both (sequence held until defined        readings are observed).    -   Harvest—harvesting of product may be a one-time individual event        (e.g., infection—infection amplification period—harvest),        multiple occurrences, or continuous, triggered sometime after        growth starts.    -   Unload—Un-mounting of the cultureware module from the instrument        module.

Computer-readable media include both volatile and nonvolatile media,removable and nonremovable media, and contemplate media readable by adatabase, a switch, and various other network devices. By way ofexample, and not limitation, computer-readable media incorporate mediaimplemented in any method or technology for storing information.Examples of stored information include computer-useable instructions,data structures, program modules, and other data representations. Mediaexamples include, but are not limited to, information-delivery media,RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM,digital versatile discs (DVD), holographic media or other optical discstorage, magnetic cassettes, magnetic tape, magnetic disk storage, andother magnetic storage devices. These technologies can store datamomentarily, temporarily, or permanently. In an embodiment,non-transitory media are used.

The invention can be practiced in distributed-computing environmentswhere tasks are performed by remote-processing devices that are linkedthrough a communications network or other communication medium. In adistributed-computing environment, program modules can be located inboth local and remote computer-storage media including memory storagedevices. The computer-useable instructions form an interface to allow acomputer to react according to a source of input. The instructionscooperate with other code segments or modules to initiate a variety oftasks in response to data received in conjunction with the source of thereceived data.

The present invention can be practiced in a network environment such asa communications network. Such networks are widely used to connectvarious types of network elements, such as routers, servers, gateways,and so forth. Further, the invention can be practiced in a multi-networkenvironment having various, connected public and/or private networks.

Communication between network elements can be wireless or wireline(wired). As will be appreciated by those skilled in the art,communication networks can take several different forms and can useseveral different communication protocols.

Embodiments of the subject invention can be embodied in a processingsystem. Components of the processing system can be housed on a singlecomputer or distributed across a network as is known in the art. In anembodiment, components of the processing system are distributed oncomputer-readable media. In an embodiment, a user can access theprocessing system via a client device. In an embodiment, some of thefunctions or the processing system can be stored and/or executed on sucha device. Such devices can take any of a variety of forms. By way ofexample, a client device may be a desktop, laptop, or tablet computer, apersonal digital assistant (PDA), an MP3 player, a communication devicesuch as a telephone, pager, email reader, or text messaging device, orany combination of these or other devices. In an embodiment, a clientdevice can connect to the processing system via a network. As discussedabove, the client device may communicate with the network using variousaccess technologies, both wireless and wireline. Moreover, the clientdevice may include one or more input and output interfaces that supportuser access to the processing system. Such user interfaces can furtherinclude various input and output devices which facilitate entry ofinformation by the user or presentation of information to the user. Suchinput and output devices can include, but are not limited to, a mouse,touch-pad, touch-screen, or other pointing device, a keyboard, a camera,a monitor, a microphone, a speaker, a printer, a scanner, among othersuch devices. As further discussed above, the client devices can supportvarious styles and types of client applications.

Thus, one aspect of the invention provides one or more computer-readablemedia having computer-useable instructions embodied thereon forperforming the method for large-scale production of cells and/orcell-derived products. In some embodiments, the steps of the method areperformed by one or more suitably programmed computers. In someembodiments, the computer executable instructions for performing one ormore of the steps of the method are provided on the one or more computerreadable media. In some embodiments, the computer executableinstructions are provided as one or more program modules, such asroutines, programs, objects, components, and/or data structures. Thecomputer-readable media are non-transitory computer readable, whichincludes all computer-readable except for a transitory propagatingsignal. Thus, a non-transitory computer readable medium includes a harddrive, compact disc, DVD, flash memory, USB drive, volatile memory, anda memory card, but does not include a transitory signal per se.Accordingly, the term “non-transitory” is not intended to excludecomputer readable media such as a volatile memory or RAM, where the datastored thereon is only temporarily stored, or stored in a “transitory”fashion.

VII. EXEMPLIFIED EMBODIMENTS

The following are exemplified embodiments.

Embodiment 1

A production module for production of cells and/or cell-derivedproducts, comprising: (a) a cultureware module comprising one or morebioreactors and an interface plate with the one or more bioreactorsmounted thereto; and (b) an instrument module comprising hardware tosupport cell culture growth, wherein said instrument module and saidcultureware module are adapted for removable attachment to one another.

Embodiment 2

The production module of embodiment 1, wherein said cultureware modulecomprises a plurality of bioreactors.

Embodiment 3

The production module of embodiment 2, wherein the plurality ofbioreactors are connected (in fluid communication) by a flow path thatpermits inoculation of one or more bioreactors in the plurality toresult in inoculation of one or more other (“downstream”) bioreactors inthe plurality.

Embodiment 4

The production module of any one of embodiments 1 to 3, wherein the oneor more bioreactors are hollow-fiber bioreactors.

Embodiment 5

A production suite comprising a plurality of production modules of anyone of embodiments 1 to 4, functionally connected.

Embodiment 6

The production suite of embodiment 5, wherein the plurality ofproduction modules are connected (in fluid communication) by a flow paththat permits inoculation of one or more production modules in theplurality of production modules to result in inoculation of one or moreother (“downstream”) production modules in the plurality of productionmodules.

Embodiment 7

The production suite of embodiment 5, further comprising a room with oneor more support surfaces for supporting the plurality of productionmodules.

Embodiment 8

The production suite of any one of embodiments 5 to 7, wherein the roomis environment-controlled (e.g., temperature controlled and/or humiditycontrolled, etc.).

Embodiment 9

The production suite of embodiment 7 or 8, wherein the room is a modularand/or relocatable building.

Embodiment 10

The production suite of embodiment 9, further comprising one or morereceivers affixed to the building frame or wall, for receiving a liftingattachment allowing transport of the building onto a truck, trailer,vessel, aircraft, or other conveyance.

Embodiment 11

The production suite of any one of embodiments 7 to 10, wherein the roomincludes environmental filtration and containment to an extent necessaryfor the cell or cell-derived product.

Embodiment 12

The production suite of any one of embodiments 7 to 11, wherein the roomhas controlled ingress and egress (e.g., to a gowning area).

Embodiment 13

The production suite of any one of embodiments 7 to 12, wherein the roomhas external control and monitoring stations to allow an operator tocheck or make adjustments in the operation of the production suite.

Embodiment 14

The production suite of any one of embodiments 7 to 13, wherein the roomfurther comprises one or more video monitoring devices for monitoringthe production modules of the production suite.

Embodiment 15

A method for large-scale production of cells and/or cell-derivedproducts, comprising providing one or more production modules or one ormore production suites of any preceding embodiment, introducing cellsinto the one or more bioreactors; culturing the cells to produce growncells and/or cell-derived products; and harvesting the grown cellsand/or cell-derived products.

Embodiment 16

The method of embodiment 15, wherein the cells are mammalian cells,insect cells, avian cells, or plant cells.

Embodiment 17

The method of embodiment 15, wherein the cells are selected from amongMadin-Darby canine kidney (MDCK) cells, African green monkey kidney(Vero) cells, human fetal retina (Per.C6) cells, duck embryonic retina(AGE1.CR) cells, murine C127 cells, 3T3 cells, COS cells, humanosteosarcoma cells, MRC-5 cells, BHK cells, CHO (Chinese hamster ovary)cells, HEK 293 cells, rHEK 293 cells, human fibroblast cells, stromacells, or hepatocytes.

Embodiment 18

The method of any one of embodiments 15 or 17, wherein the cell-derivedproducts are selected from among immunoglobulins, proteins, virus, andvirus-like particles.

Embodiment 19

One or more computer-readable media having computer-useable instructionsembodied thereon for performing the method of any one of embodiments15-18.

Embodiment 20

The media of embodiment 19, wherein one or more of the steps of themethod are performed by one or more suitably programmed computers.

Embodiment 21

The media of embodiment 20, wherein computer executable instructions forperforming one or more of the steps of the method are provided on theone or more computer readable media.

Embodiment 22

The media of embodiment 21, wherein the computer executable instructionsare provided as one or more program modules.

Embodiment 23

The media of embodiment 22, wherein the program modules includeroutines, programs, objects, components, and/or data structures.

Embodiment 24

The media of embodiment of any one of embodiments 19 to 23, wherein themedia includes one, two, three, four, five, six, seven, eight, or nineof the following modes: idle, load, acceptance testing, flush,pre-inoculation, inoculation, growth, harvest, and unload.

Embodiment 25

The media of embodiment 24, wherein the media includes each of thefollowing modes: idle, load, acceptance testing, flush, pre-inoculation,inoculation, growth, harvest, and unload.

VIII. DEFINITIONS

In order to more clearly and concisely describe the subject matter ofthe claims, the following definitions are intended to provide guidanceas to the meaning of specific terms used herein.

It is to be noted that the singular forms “a,” “an,” and “the” as usedherein include “at least one” and “one or more” unless stated otherwise.Thus, for example, reference to “a cultureware module” is inclusive ofmore than one cultureware module, reference to “a bioreactor” isinclusive of more than one bioreactor, and the like.

As used herein, the terms “comprising”, “consisting of” and “consistingessentially of” are defined according to their standard meaning. Theterms may be substituted for one another herein in order to attach thespecific meaning associated with each term.

The term “virus,” is used interchangeably herein with fragment orportion of viruses (e.g., virus-like particles (VLPs)). Thus, the term“virus” is inclusive of viruses and viral particles. Any virus maypotentially be produced and purified using the bioreactor, apparatus,and methods of the invention. The selection of virus is only limited bythe availability of a suitable host cell for production. Virus may belytic virus or budding virus. Virus may be enveloped or naked virus. Forexample, orthomyxovirus (e.g., influenza virus A, B, and C),paramyxovirus (e.g., measles, respiratory syncytial virus), arbovirus(e.g., Dengue virus), filovirus (e.g., Ebola), enterovirus (e.g., poliovirus), rhinovirus, herpes virus, and hepadina virus can be produced andpurified. In some embodiments, the virus is a non-human animal virus. Insome embodiments, the virus is a human virus, such as human influenzavirus (e.g., strains H5N1 and H1N1).

As used herein, the terms “automation” and “automated” are usedinterchangeably and refer to the controlled operation of an apparatus,process, or system by mechanical or electronic devices. Automatedmethods of the invention include sequential, pre-determined steps, whichare internally controlled by software driven servo-actuators. Thus, themethods are standardized, efficient, and free of human error.

As used herein, the term “computer system” generally includes one ormore computers, peripheral equipment, and software that perform dataprocessing. A “user” or “operator” in general includes a person thatutilizes the apparatus of the invention such as through a userinterface. A “computer” is generally a functional unit that can performsubstantial computations, including numerous arithmetic operations andlogic operations without human intervention.

As used herein, the term “culture” is used to denote the maintenance orcultivation of cells in vitro including the culture of single cells.Cultures can be cell, tissue, or organ cultures, depending upon theextent of organization. Cells may be cultured in a bioreactor. Theintended product of the cell culture may be the cells themselves, orbiomolecules produced by the cells, or virus, VLPs, or viral vectorsproduced by the cells, or a combination of two or more of the foregoing.

As used herein, “cultureware” refers to components which come in contactwith cell or cell-derived product-containing medium, the purified orunpurified product, or any liquid involved in the cell culture and/orpurification process.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

We claim:
 1. A method for large-scale production of cells and/orcell-derived products, comprising: providing one or more productionmodules, wherein each of the one or more production modules comprises:(a) a cultureware module comprising a plurality of hollow-fiberbioreactors, an intracapillary reservoir, an extracapillary reservoir,and an interface plate with said plurality of hollow-fiber bioreactorsmounted thereto, wherein each hollow-fiber bioreactor has anintracapillary space and an extracapillary space, and wherein eachhollow-fiber bioreactor is connected to an adjacent hollow-fiberbioreactor by a flow path; and an instrument module comprising a pumpfor circulating cell culture medium through the plurality ofhollow-fiber bioreactors, an intracapillary manifold connected to theintracapillary space and the intracapillary reservoir, an extracapillarymanifold connected to the extracapillary space and the extracapillaryreservoir, wherein the instrument module and the cultureware module areadapted for removable attachment to one another; introducing cells intoone or more of the hollow-fiber bioreactors; culturing the cells toproduce grown cells and/or cell-derived products; and harvesting thegrown cells and/or cell-derived products.
 2. The method of claim 1,wherein the cells are mammalian cells, insect cells, avian cells, orplant cells.
 3. The method of claim 1, wherein the cells are selectedfrom among Madin-Darby canine kidney (MDCK) cells, African green monkeykidney (Vero) cells, human fetal retina (Per.C6) cells, duck embryonicretina (AGE1.CR) cells, murine C127 cells, 3T3 cells, COS cells, humanosteosarcoma cells, MRC-5 cells, BHK cells, CHO (Chinese hamster ovary)cells, HEK 293 cells, rHEK 293 cells, human fibroblast cells, stromacells, or hepatocytes.
 4. The method of claim 1, wherein thecell-derived products are selected from among immunoglobulins, proteins,virus, and virus-like particles.
 5. The method of claim 1, wherein saidproviding one or more production modules comprises providing one or moreproduction suites, wherein the production suite comprises a plurality ofproduction modules, and wherein each production module of eachproduction suite is functionally connected.
 6. The method of claim 5,further comprising providing a room with one or more support surfacesfor supporting the plurality of production modules.
 7. The method ofclaim 6, wherein the room is environment-controlled.
 8. The method ofclaim 6, wherein the room includes environmental filtration andcontainment to an extent necessary for the cell or cell-derived product.9. The method of claim 6, wherein the room is a modular and/orrelocatable building.
 10. The method of claim 9, wherein the modularand/or relocatable building further comprises one or more receiversaffixed to the building frame or wall, for receiving a liftingattachment, allowing transport of the building onto a truck, trailer,vessel, aircraft, or other conveyance.
 11. The method of claim 6,wherein the room has external control and monitoring stations to allowan operator to check or make adjustments in the operation of theproduction suite.
 12. The method of claim 6, wherein the room furthercomprises one or more video monitoring devices for monitoring theproduction modules of the production suite.
 13. The method of claim 6,wherein the room has controlled ingress and egress.
 14. The method ofclaim 1, wherein the flow path connecting adjacent hollow-fiberbioreactor permits inoculation of one or more hollow-fiber bioreactorsin the plurality to result in inoculation of one or more otherhollow-fiber bioreactors in the plurality.